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Upregulation of GADD45 α in light-damaged retinal pigment epithelial cells

View Article: PubMed Central - PubMed

ABSTRACT

To better understand the molecular mechanisms responsible for light-induced damage in retinal pigmented epithelial (RPE) cells, we developed an automated device to recapitulate intense light exposure. When compared with human fibroblasts, ARPE-19 cells that had been exposed to blue-rich light-emitting diode-light of 10 000 Lux at 37 °C for 9 h displayed dramatic cellular apoptosis. Collectively, gene expression profiling and qPCR demonstrated that growth arrest and DNA damage-45α (GADD45α) expression was markedly upregulated. Transient knockdown of GADD45α partially attenuated light-damage-induced apoptosis in ARPE-19 cells, whereas GADD45α overexpression dramatically increased it. These results demonstrate the critical function of GADD45α in light-induced RPE cellular apoptosis. Quantitative reverse transcription-PCR and western blotting revealed that the upregulation of GADD45α was under direct control of p53. Moreover, treatment with Ly294002, an inhibitor of AKT phosphorylation, further promoted GADD45α gene transcription in both non-light and light-damaged ARPE-19 cells. Treatment also exacerbated RPE cellular apoptosis after light exposure, confirming that inhibition of Akt phosphorylation increases GADD45α expression. Collectively, our findings reveal that light irrigation induces human RPE cellular apoptosis through upregulation of GADD45α expression mediated through both the p53 and phosphatidylinositol 3-kinase-AKT signaling pathways. These results provide new insights into human retinal diseases elicited by light damage and open a new avenue for disease prevention and treatment.

No MeSH data available.


GADD45α stimulates apoptosis in ARPE-19 cells. (a) Overexpression of GADD45α in ARPE-19 cells resulted in a dose-dependent decrease in cell viability. Each point represents the mean value±S.E.M. (n=4, *P<0.05, ***P<0.001). (b) Overexpression of GADD45α significantly decreased cell viability, either with or without light damage. Each point represents the mean value±S.E.M. (n=4, ***P<0.001). (c) Knockdown of GADD45α in ARPE-19 cells using lentiviral delivery of shRNA. Results are given as mean value±S.E.M. (n=3, ***P<0.001). (d) Silencing GADD45α significantly reversed apoptosis in light-damaged RPE cells. Each value represents the mean value±S.E.M. (n=4, **P<0.01).
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fig4: GADD45α stimulates apoptosis in ARPE-19 cells. (a) Overexpression of GADD45α in ARPE-19 cells resulted in a dose-dependent decrease in cell viability. Each point represents the mean value±S.E.M. (n=4, *P<0.05, ***P<0.001). (b) Overexpression of GADD45α significantly decreased cell viability, either with or without light damage. Each point represents the mean value±S.E.M. (n=4, ***P<0.001). (c) Knockdown of GADD45α in ARPE-19 cells using lentiviral delivery of shRNA. Results are given as mean value±S.E.M. (n=3, ***P<0.001). (d) Silencing GADD45α significantly reversed apoptosis in light-damaged RPE cells. Each value represents the mean value±S.E.M. (n=4, **P<0.01).

Mentions: The next question we asked was whether GADD45α upregulation causally resulted in ARPE-19 apoptosis. To address this, we investigated the role of GADD45α in ARPE-19 cells. First, overexpression of GADD45α led to a significant dose-dependent decline of cell viability when compared with vehicle control (Figure 4a). Furthermore, forced expression of GADD45α markedly exacerbated cell death in light-exposed ARPE-19 cells (Figure 4b). To further elucidate the biological effect of GADD45α in light-induced apoptosis, we silenced GADD45α in ARPE-19 cells using lentiviral delivery of short hairpin RNAs (shRNAs; Figure 4c). Knockdown of GADD45α significantly reversed the previously observed cell viability reduction in light-damaged ARPE-19 cells (Figure 4d). Since the decrease in cell viability was primarily caused by apoptosis, these data strongly suggest that GADD45α is an important regulator of light-induced apoptosis of RPE cells in vitro.


Upregulation of GADD45 α in light-damaged retinal pigment epithelial cells
GADD45α stimulates apoptosis in ARPE-19 cells. (a) Overexpression of GADD45α in ARPE-19 cells resulted in a dose-dependent decrease in cell viability. Each point represents the mean value±S.E.M. (n=4, *P<0.05, ***P<0.001). (b) Overexpression of GADD45α significantly decreased cell viability, either with or without light damage. Each point represents the mean value±S.E.M. (n=4, ***P<0.001). (c) Knockdown of GADD45α in ARPE-19 cells using lentiviral delivery of shRNA. Results are given as mean value±S.E.M. (n=3, ***P<0.001). (d) Silencing GADD45α significantly reversed apoptosis in light-damaged RPE cells. Each value represents the mean value±S.E.M. (n=4, **P<0.01).
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fig4: GADD45α stimulates apoptosis in ARPE-19 cells. (a) Overexpression of GADD45α in ARPE-19 cells resulted in a dose-dependent decrease in cell viability. Each point represents the mean value±S.E.M. (n=4, *P<0.05, ***P<0.001). (b) Overexpression of GADD45α significantly decreased cell viability, either with or without light damage. Each point represents the mean value±S.E.M. (n=4, ***P<0.001). (c) Knockdown of GADD45α in ARPE-19 cells using lentiviral delivery of shRNA. Results are given as mean value±S.E.M. (n=3, ***P<0.001). (d) Silencing GADD45α significantly reversed apoptosis in light-damaged RPE cells. Each value represents the mean value±S.E.M. (n=4, **P<0.01).
Mentions: The next question we asked was whether GADD45α upregulation causally resulted in ARPE-19 apoptosis. To address this, we investigated the role of GADD45α in ARPE-19 cells. First, overexpression of GADD45α led to a significant dose-dependent decline of cell viability when compared with vehicle control (Figure 4a). Furthermore, forced expression of GADD45α markedly exacerbated cell death in light-exposed ARPE-19 cells (Figure 4b). To further elucidate the biological effect of GADD45α in light-induced apoptosis, we silenced GADD45α in ARPE-19 cells using lentiviral delivery of short hairpin RNAs (shRNAs; Figure 4c). Knockdown of GADD45α significantly reversed the previously observed cell viability reduction in light-damaged ARPE-19 cells (Figure 4d). Since the decrease in cell viability was primarily caused by apoptosis, these data strongly suggest that GADD45α is an important regulator of light-induced apoptosis of RPE cells in vitro.

View Article: PubMed Central - PubMed

ABSTRACT

To better understand the molecular mechanisms responsible for light-induced damage in retinal pigmented epithelial (RPE) cells, we developed an automated device to recapitulate intense light exposure. When compared with human fibroblasts, ARPE-19 cells that had been exposed to blue-rich light-emitting diode-light of 10&thinsp;000&thinsp;Lux at 37&thinsp;&deg;C for 9&thinsp;h displayed dramatic cellular apoptosis. Collectively, gene expression profiling and qPCR demonstrated that growth arrest and DNA damage-45&alpha; (GADD45&alpha;) expression was markedly upregulated. Transient knockdown of GADD45&alpha; partially attenuated light-damage-induced apoptosis in ARPE-19 cells, whereas GADD45&alpha; overexpression dramatically increased it. These results demonstrate the critical function of GADD45&alpha; in light-induced RPE cellular apoptosis. Quantitative reverse transcription-PCR and western blotting revealed that the upregulation of GADD45&alpha; was under direct control of p53. Moreover, treatment with Ly294002, an inhibitor of AKT phosphorylation, further promoted GADD45&alpha; gene transcription in both non-light and light-damaged ARPE-19 cells. Treatment also exacerbated RPE cellular apoptosis after light exposure, confirming that inhibition of Akt phosphorylation increases GADD45&alpha; expression. Collectively, our findings reveal that light irrigation induces human RPE cellular apoptosis through upregulation of GADD45&alpha; expression mediated through both the p53 and phosphatidylinositol 3-kinase-AKT signaling pathways. These results provide new insights into human retinal diseases elicited by light damage and open a new avenue for disease prevention and treatment.

No MeSH data available.